1. Field of the Invention
This invention relates generally to the protein degradation pathways, and more specifically to compositions and methods for modulating the isopeptidase activity of the COP9 signalsome and, therefore, ubiquitin dependent protein degradation.
2. Background Information
The function of a protein is regulated via various means in a cell. One way to regulate protein function is via conjugation and deconjugation of a modifier protein to a target protein, e.g., neddylation and deneddylation or ubiquitination and deubiquitination.
The major route for protein degradation in the nucleus and cytoplasm of eukaryotic cells is via the ubiquitin/26S proteasome pathway. The 26S proteasome comprises two major subparticles: the 20S proteasome and the 19S regulatory particle. The 20S proteasome is a cylindrical structure with an internal cavity that contains the peptidase active sites. Substrates of the proteasome are inserted into the cylinder, where they are susceptible to digestion by the peptidase active sites of the 20S proteasome. Entry into the 20S proteasome cylinder is governed by the 19S regulatory particle, which caps the ends of the 20S cylinder.
The 19S regulatory particle binds ubiquitinated substrates and translocates them into the inner cavity of the 20S cylinder, where they are degraded. The 19S regulatory particle can be further subdivided into two multiprotein complexes: the base and the lid. The base comprises a set of six ATPases that are thought to unfold substrates and translocate them into the 20S proteasome. The lid is comprised of a set of eight proteins of unknown function. Biochemical data indicate that the presence of the lid renders the proteasome selective for degrading ubiquitinated proteins, but the basis for this selectivity is not known.
The lid subcomplex of the 26S proteasome is evolutionarily related to the COP9-signalsome (CSN) complex, but the significance of this similarity has remained unknown. There are reports that 26S proteasome preparations contain a variety of associated ubiquitin isopeptidase activities (Eytan et al., J. Biol. Chem. 268:4668-74, 1993; Verma et al., Mol. Biol. Cell 11:3425, 2000), but it has not been demonstrated that an ubiquitinated substrate can be completely deubiquitinated by purified 26S proteasome to yield unmodified substrate. The failure to detect such a reaction product may be due to a tight coupling between the deubiquitination of a substrate and its subsequent degradation within the internal cavity of the 20S proteasome.
Proteins that are destined for degradation by the ubiquitin/26S pathway are marked by the attachment of a polyubiquitin chain to the side chains of lysine residues on the target protein. The ubiquitinated protein is recognized by the 26S proteasome by a mechanism that remains poorly understood. Subsequently the ubiquitinated protein is disengaged from any tightly bound partners, unfolded, and translocated into the central cavity of the 20S complex, where it is exhaustively degraded by the proteolytic active sites that are present in this inner cavity.
Despite many years of intensive study of this system, it remains unclear what happens to the substrate-bound polyubiquitin chains that target the substrate for degradation. It appears that ubiquitin is not degraded by the proteasome but, in fact, is recycled. However it remains unclear if the ubiquitin chains enter the inner cavity of the proteasome and emerge unscathed, or are cleaved from the substrate protein prior to or during its translocation into the inner cavity of the 20S. In prior work (Eytan et al., supra, 1993), it was demonstrated that there is an isopeptidase activity or activities associated with the intact 26S proteasome that is able to release free ubiquitin monomers from ubiquitinated substrates that are degraded by the 26S proteasome. It was demonstrated that this activity is sensitive to the metal ion chelator 1,10-phenanthroline, but the identity of a polypeptide that harbors this activity was not established, nor was it established that this activity is intrinsic to the 26S proteasome as opposed to being intrinsic to a protein that binds transiently to the 26S proteasome. This prior work also failed to disclose whether the ubiquitin isopeptidase activity is critical to the protein-degrading function of the 26S proteasome.
Nedd8 is an ubiquitin-like protein that is covalently linked via an isopeptide bond between its carboxy terminus and the epsilon amino group of lysine residues in target proteins (referred to as neddylation). The attachment of Nedd8 to target proteins requires the combined action of a Nedd8-activating enzyme composed of Ula1 and Uba3 subunits (analogous to ubiquitin-activating enzyme, E1), and Ubc12, which is homologous to the ubiquitin-conjugating enzymes (E2). There is no known requirement for an activity equivalent to the ubiquitin ligase (E3) component of ubiquitination pathways. The Nedd8 modification, like ubiquitination, is likely dynamic, although little is known about the nature of the enzymes that would cleave Nedd8 from its targets (i.e., deneddylate, also referred to as deneddylation or deneddylating activity).
A ubiquitin isopeptidase (USP21) can deneddylate Cul1-Nedd8 conjugates, and a second enzyme UCH-L3, can cleave Nedd8-containing fusion proteins at the C-terminus of Nedd8 to release mature Nedd8; Nedd8, like ubiquitin, is made as a precursor with additional C-terminal residues that must be removed before it can be conjugated to proteins. Despite the fact that both USP21 and UCH-L3 can metabolize Nedd8-based substrates, they also act on ubiquitin-based substrates, and it remains unclear whether their biochemical activity towards Nedd8 is relevant in the context of a cell.
In contrast to ubiquitin, the attachment of Nedd8 to proteins does not mark them for degradation. Rather, it appears that neddylation acts to modify protein function, much like phosphorylation. The cullins are the only proteins that have been found to be conjugated with Nedd8. The cullins are a family of six related proteins that bind a RING-H2 domain protein to form the catalytic core of multisubunit ubiquitin ligases. All cullins examined are modified by attachment of Nedd8, and neddylation of Cul1 potentiates the ubiquitin ligase of the SCF complex within which Cul1 resides. Thus, for Cul1-based ubiquitin ligases, neddylation serves as a positive regulator of activity. However, for other cullin-based ubiquitin ligases, the role, if any, of neddylation remains uncertain.
The COP9/signalsome (“CSN”) was originally identified as a regulator of photomorphogenetic development in plants. In seedlings grown in the dark, CSN enables a putative ubiquitin ligase, COP1, to mediate rapid turnover of the transcriptional regulatory protein HY5 in the nucleus. In seedlings that have been exposed to light or in CSN mutants, COP1 redistributes from the nucleus to the cytoplasm, thereby stabilizing HY5 and allowing it to accumulate in the nucleus. HY5, in turn, activates the transcription of a broad palette of genes that mediate photomorphogenetic development. Although the general physiological role of CSN in photomorphogenesis has been defined, little is known about other potential physiological functions for CSN, and the biochemical function of CSN remains elusive. Based on the observation that the eight subunits of CSN share homology to subunits of the lid subcomplex of the 19S regulator of the 26S proteasome, it has been suggested that CSN may play a role in ubiquitin-dependent proteolysis. However, a similar pattern of homology is shared with subunits of the eukaryotic initiation factor-3 (eIF3) complex. Besides plants, CSN complexes have been discovered in human cells, Drosophila, and the fission yeast Schizosaccharomyces pombe. Surprisingly, the budding yeast Saccharomyces cerevisiae does not contain an apparent CSN complex, although it contains a gene homologous to the CSN5/JAB1 subunit of CSN complex, thus implicating CSN5 as a critical component of CSN.
Given the role of protein degradation in maintaining normal cell function, and the disruption of this pathway in association with various disorders such as cancer, an understanding of the components involved in regulating ubiquitination and protein degradation would allow the development of drugs that can modify protein degradation. Thus, a need exists to identify the active domain and site of peptidase activity, particularly isopeptidase activity of a protein involved in deconjugation of a modifier protein from a target protein, e.g., deneddylation or deubiquitination such that screening assays can be developed to identify drugs that can selectively modulate protein modification and degradation. The present invention satisfies this need and provides additional advantages.